Regenerative and Technological Section / Mini-Review

Received: June 12, 2009

Accepted: February 5, 2010
Published online: May 6, 2010

Gerontology 2011;57:8594
DOI: 10.1159/000314530

Dental Tissue Regeneration

A Mini-Review
A.-H.Yen P.C.Yelick
Department of Oral and Maxillofacial Pathology, Division of Craniofacial and Molecular Genetics, Tufts University,
Boston, Mass., USA

Abstract
Background: With todays 21st century technological advancements, it is expected that individuals will either retain
their natural teeth or obtain functional tooth replacements
throughout their entire life. Modern dental therapies for the
replacement of missing teeth largely utilize partial or complete dentures and titanium implants capped with prosthetic crowns. Although these prostheses serve a purpose, they
are not equivalent, neither in function nor aesthetics, to natural teeth. Recent progress in dental tissue engineering has
lent significant credibility to the concept that biological replacement teeth therapies may soon be available to replace
missing teeth. Objective: In this review, we summarize the
emerging concepts of whole-tooth replacement strategies,
using postnatal dental stem cells (DSCs) and dental tissue
engineering approaches. Methods: We provide a thorough
and extensive review of the literature. Results: Current approaches to achieve clinically relevant biological replacement tooth therapies rely on the cultivation of DSCs capable
of relaying odontogenic induction signals, through dental
epithelial-mesenchymal cell interactions. DSC expansion
and differentiation can be achieved by programming progenitor stem cells to adopt dental lineages, using instructive,

2010 S. Karger AG, Basel

0304324X/11/05710085$38.00/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com

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bioengineered scaffold materials. Periodontal ligament regeneration in particular has demonstrated significant progress recently, despite the somewhat unpredictable clinical
outcomes, with regard to its capacity to augment conventional metallic dental implants and as an important component for whole-tooth tissue engineering. Following recent
advances made in DSC and tissue engineering research, various research groups are in the midst of performing proof of
principle experiments for whole-tooth regeneration, with
associated functional periodontal tissues. This mini-review
focuses on recent and promising developments in the fields
of pulp and periodontal tissue DSCs that are of particular relevance for dental tissue and whole-tooth regeneration. Conclusion: Continued advances in the derivation of useable
DSC populations and optimally designed scaffold materials
unequivocally support the feasibility of dental tissue and
whole-tooth tissue engineering.
Copyright 2010 S. Karger AG, Basel

Introduction

Tooth loss is an early indicator of accelerated aging.

According to a recent study, persons who were edentulous at age 70 had a significantly higher risk of mortality
21 years later [1]. Although continental European and
Scandinavian countries such as Sweden have exhibited

Dr. Pamela C. Yelick

Department of Oral and Maxillofacial Pathology
Division of Craniofacial and Molecular Genetics, Tufts University
Boston, MA 02111 (USA)
Tel. +1 617 636 2430, Fax +1 617 636 2432, E-Mail pamela.yelick@tufts.edu

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Key Words
Teeth Stem cells Regeneration Tissue engineering

86

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velopment, at the dental lamina stage, and is regulated by

several homeobox genes, such as Msx1, which are specifically expressed in the predental mesenchyme. The
physical morphological process of tooth development begins at the cap stage and is coordinated by enamel knot
signaling centers, transient structures present in the dental epithelium thought to be regulated by homeobox gene
expression and which mark tooth cusp formation [8].
Secondary, tertiary and quaternary enamel knots sequentially appear and form the pattern of tooth crowns.
By the late cap stage, the enamel knots will have disappeared by apoptosis.
Having received early odontogenic signals from the
dental epithelium, the ectomesenchyme next becomes
the principal source of signaling for subsequent tooth development, directing the dental epithelium to differentiate into enamel-secreting ameloblasts and the adjacent
mesenchyme to differentiate into dentin-secreting odontoblasts [9]. At the bell stage, there is a recognizable tooth
germ consisting of an organized enamel organ, dental papilla and dental follicle. The enamel organ, responsible
for enamel formation, consists of inner and outer enamel
epithelium, stratum intermedium and stellate reticulum.
Ameloblasts, which are formed from the inner dental epithelium, differentiate to produce enamel, which is composed of more than 90% hydroxyapatite and is known to
be the hardest tissue in the body. The cervical region of
the inner and outer enamel epithelium gives rise to Hertwigs epithelial root sheath, a rudimentary tooth root
structure that initiates radicular dentin formation and
determines the root shape [10]. The dental papilla will
give rise to pulp tissue, a complex, vital connective tissue
composed of fibroblasts, blood vessels, nerves, lymphatic
ducts and odontoblasts. Differentiated odontoblasts, cells
derived from the mesenchymal cells directly subjacent to
and induced by the inner enamel epithelium, are terminally differentiated, postmitotic cells that have withdrawn from the cell cycle and therefore cannot proliferate
to replace irreversibly injured odontoblasts [11]. Functional odontoblasts exhibit a polarized columnar morphology that shifts into a resting state, becoming smaller
and flatter after primary dentin formation. However,
odontoblasts remain functional throughout their life and
can produce secondary dentin in response to mild trauma. The dental follicle, a transient structure during tooth
morphogenesis, eventually forms 3 major types of cells,
i.e. cementoblasts, osteoblasts and fibroblasts [12]. Cementoblasts secrete cementum on the tooth root surface,
while osteoblasts produce alveolar bone around the tooth
roots. Collagen-producing fibroblasts give rise to periYen/Yelick

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reduced edentulism, from 51% in the 1901/1902 birth cohort to 16% in the 1922 cohort [2], statistical predictions
suggest that in the USA alone, of approximately 150 million adults, over 10 million new cases of edentulism will
occur in the next decade. Edentulism not only results in
reduced oral and social functions, but also remains a major public health issue [3].
Traditional methods to treat edentulism include complete denture therapy, which is associated with complications such as denture-induced stomatitis, soft tissue hyperplasia, traumatic ulcers, altered taste perception and
burning mouth syndrome [1]. It is also well known that
tooth loss leads to alveolar bone resorption [4]. Therefore,
the need for alternative tooth replacement therapies is
quite evident. The concept of osseointegration was introduced in the 1950s by Per-Ingvar Branemark, who observed the direct structural and functional bond formation between bone and titanium. Nowadays, endosseous
implants are a commonly accepted treatment option. Titanium was subsequently regarded as a durable and biocompatible dental implant material which allowed the
functional replacement of missing teeth. Since the 1950s,
considerable improvements have been made to increase
osseointegration of titanium implants, including surface
modifications to improve the mechanical, physical and
chemical characteristics of the implant [5]. Recently, biomimetic modification of the titanium implant surface
has been proposed as a novel approach to further improve
osseointegration [6]. However, osseointegration represents a direct connection between the implant and bone
tissue and lacks the periodontium and cementum tissues
present in naturally formed teeth, which function to
cushion and modulate the mechanical stress of mastication. Therefore, strategies to generate dental implants
with associated periodontal tissues have become a new
approach in tooth replacement therapies [7].
A logical strategy for whole-tooth tissue engineering
is to mimic natural tooth development, which is first evident as a localized thickening of the dental epithelium,
the dental lamina, which subsequently invaginates into
underlying neural crest-derived ectomesenchyme to
form a bud. At the tooth initiation stage, odontogenic signals initiated in the inner dental epithelium stimulate
proliferation and condensation of subjacent dental mesenchyme. Fibroblast growth factor-8, bone morphogenetic protein-4, Shh and Wnt10b, expressed by the dental
epithelium, induce and regulate the expression of downstream transcription factors in the ectomesenchyme, including Barx1, Dlx1/2, Lhx6, Lhx7, Msx1, Pax9, Ptc and
Lef1. Tooth shape specification occurs early in tooth de-

epithelial cells

mesenchymal
cells

od

e
am

si

Facilitateearly dental
epithelialand
mesenchymalcell
interactions

sr

Enamel

Dentin

todirectlater
mineralizeddental
tissueformation.
Cementum

odontal ligament (PDL) tissues, which attach tooth roots

to the surrounding alveolar bone through Sharpeys fibers extending into the cementum layer. The PDL functions as a mechanical cushion for masticatory forces, is
an organ of proprioception via embedded nerve cells and
is regarded as the main impetus for the tooth eruption
process. The complex structure consisting of the PDL,
adjacent cementum and surrounding alveolar bone is
called the periodontium. After the eruption of a tooth
into the oral cavity, the tooth is clinically divided into 2
parts, i.e. the crown and the root. Crowns are the visible
structures in the oral cavity, and roots are the embedded
tooth regions that connect the surrounding alveolar bone
with the cementum, anchoring the tooth in place. Ana-

tomically, the enamel-surfaced tooth extends to the cemento-enamel junction, while the cementum-covered
tooth roots are present below the cemento-enamel junction. At the present time, there is extensive knowledge of
tooth crown development, while relatively little is known
about the molecular signaling mechanisms regulating
tooth root development.
Based on these intricate and tightly regulated tooth
developmental processes, as described above, dental tissue engineering approaches are currently being utilized
to generate functional bioengineered replacement teeth
and dental tissues that closely match the physical and mechanical properties of naturally formed tooth tissues, as
schematized below (fig.1).

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Fig. 1. Whole-tooth tissue engineering. od = Odontoblasts; d = dentin; e = enamel; am = ameloblasts; si = stratum intermedium; sr =stellate reticulum.

Partial reparative tooth tissues had been created prior

to the conception of tissue engineering [13]. For example,
dentin production was shown to be induced by calcium
hydroxide in pulp-capping procedures in the 1980s [14],
although the underlying mechanisms remain elusive.
Guided tissue regeneration was successfully used to regenerate periodontal tissues and had become a successful
and widely available clinical therapy [15]. The emergence
of whole-tooth tissue engineering stemmed from the demand for biologically based dental tissue and wholetooth replacements and was made possible by the marriage of biological, developmental and material sciences
[13]. The concept underlying tissue engineering is to use
ex vivo expanded progenitor cell populations, or stem
cells, seeded into and grown within supporting biocompatible materials implanted in an appropriate environment, to create functional tissue replacements.
Stem cells are defined as clonogenic cells exhibiting
the capacity for self-renewal and multilineage differentiation. Stem cells can be divided into 2 main types, i.e.
embryonic stem cells (ESCs) and adult stem cells. Pluripotent ESCs are derived from the inner cell mass of
mammalian blastocysts and can be maintained indefinitely in culture [16]. The demonstrated conversion of
mouse ESCs into differentiated neurons in an animal
model of Parkinsons disease, and into islet cells in diabetes, together with successful isolation and characterization of human ESCs, has stimulated vigorous interest in
their potential use in clinically relevant applications in
humans. Human ESCs promise a renewable source of
progenitor cells that can be induced to differentiate into
precursors of virtually any cell type. However, enthusiasm regarding ESC use is dampened by concerns regarding possible tumorigenic and malignant properties potentially exhibited by undifferentiated ESCs when implanted into ectopic sites, along with polarizing ethical
issues regarding the use of human ESCs.
Until very recently it was generally assumed that stem
cells in adult tissues were limited to specific cell fates.
However, adult stem cells harvested from bone marrow
[17], hematopoietic [18], neuronal [19] and mesenchymal
tissues [20] have been demonstrated to differentiate into
cell types that are derived from multiple germ layers, a
feature defined as plasticity. The plasticity of adult stem
cells remains a controversial issue, due to the current lack
of adequate numbers of definitive differentiation markers, as well as a lack of reliably reproducible protocols and
results. However, adult somatic cells infected with retro88

Gerontology 2011;57:8594

viral or lentiviral vectors expressing the transcription

factors Oct4 and Sox2, along with either Klf4 and c-Myc,
or Nanog and Lin-28, exhibit an ESC-like phenotype and
have been named induced pluripotent stem (iPS) cells
[2124]. iPS cells behave similarly to ESCs, exhibiting the
capacity to differentiate into virtually any cell type, potentially providing unlimited opportunities for tissue regeneration, while avoiding ethical issues surrounding the
use of human embryos. Although issues remain regarding the possible tumorigenic potential of retrovirally
transfected cells, improved viral-free transfection methods are currently being investigated to eliminate this concern [25]. Thus, the creation of iPS cells presents potentially tremendous opportunities for the development of
patient-specific therapies.
Dental Pulp Stem Cells
The ability of human teeth to form reparative dentin
in response to deep caries and mild trauma suggests that
progenitor cells present in fully developed tooth pulp retain the ability to form functional odontoblasts, which
can produce dentin-like hard tissues [26]. The theory is
that undifferentiated mesenchymal progenitor cells existing in the dental pulp have the ability to differentiate
into odontoblast-like cells and form new dentin in response to dental injury [27]. To better understand tooth
regeneration capabilities, several populations of dental
stem cells (DSCs) have been identified and characterized. Human dental pulp cells, derived from developing
third molars and cultured in mineralization-enhancing
conditions, were demonstrated to form odontoblast-like
cells that produce dentin and also express the neuronal
marker nestin [27]. Similar studies showed that human
dental pulp stem cells (DPSCs) [28, 29] and stem cells
harvested from human exfoliated deciduous teeth
(SHED) [30] could be derived from adult and deciduous
dental pulp, respectively, as indicated by their high proliferation and colony-forming ability in culture. Besides
exhibiting the capacity to form mineralized tissues,
DPSCs and SHED can express neural markers and also
have the potential to differentiate into adipocytes [30].
When DPSCs were implanted subcutaneously into immunocompromised mice, dentin-pulp-like complexes
formed, but lamellar bone did not. Dentin and bone formation, but not dentin-pulp complexes or surrounding
alveolar bone, was observed in transplanted SHED in
vivo. Although DPSCs and SHED appear to contain
stem cells, they are also likely to contain heterogeneous
populations of differentiating pulp cells, which further
complicates analyses of these cells.
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Dental Stem Cells and Tooth Tissue Engineering

Dental Follicle Stem Cells

The dental follicle has long been considered a multipotent tissue, based on its ability to generate cementum,
bone and PDL from the ectomesenchyme-derived fibrous
tissue. Bovine dental follicle cells exhibit the ability to
form cementoblasts when transplanted into immunodeficient mice. Human dental follicle progenitor cells obtained from human third molars exhibit a characteristic
ability to attach to tissue culture plastic, express differentiated tissue makers, including nestin and Notch-1, and
to differentiate into PDL-like structures, including bone
and cementum [3640]. Further analysis revealed the
heterogeneous nature of cell populations in developing
dental follicles, through analysis of mineralization capacities in vitro and characterization of growth factor and
matrix protein gene expression patterns from several
clonally derived dental follicle cell lines cultured using
identical conditions. DSCs were identified in the dental
follicles of molars by Hoechst staining, alkaline phosphatase staining, the expression of side population stem cell
markers and the demonstrated ability to differentiate
into not only osteoblasts/cementoblasts but also adipocytes and neurons.

Periodontal Ligament Stem Cells

In common with dental pulp, periodontal ligament
(PDL) retains limited capacity to regenerate in response
to mild trauma. Multipotent progenitors from human
PDL have been identified and validated using methods
similar to those used to characterize DPSCs/SHED, including single-colony selection and magnetic activated
cell sorting with STRO-1, and PDL stem cells (PDLSCs)
have been characterized as STRO-1 and CD146/MUC18
positive [36]. Human PDLSCs display cell surface marker
characteristics and differentiation potential similar to
bone marrow stromal stem cells and DPSCs. Under defined culture conditions, PDLSCs are multipotent and
exhibit the ability to differentiate into cementoblast-like
cells, adipocytes and fibroblasts [37]. After PDLSCs were
transplanted into immunocompromised mice, cementum/PDL-like structures were formed. Human PDLSCs
expanded ex vivo and seeded in 3-dimensional scaffolds
(fibrin sponge, bovine-derived substitutes) were shown to
generate bone [38]. These cells have also been shown to
retain stem cell properties and tissue regeneration capac-

Dental Epithelial Stem Cells

Oral ectoderm-derived ameloblasts are unable to proliferate or regenerate once they have reached the maturation stage of development. Continuously growing mouse
incisors, and molars in some mammalian species, exhibit constantly replenishing populations of enamel organ
tissue-derived stellate reticulum, stratum intermedium
and surrounding outer enamel epithelial cells, providing
a source of tissues to harvest for characterization of dental epithelial stem cells and analyses of dental epithelial
tissue.
Continuously erupting mouse incisors exhibit an epithelial DSC niche located at their labial apical end, known
as the cervical loop, located at the junction of the inner
enamel epithelium and the outer enamel epithelium at
the apex of the enamel organ. The cervical loop is considered to be a determinative region regulating odontogenesis, based on its ability to produce both enamel and dentin. Early morphological observations revealed that most
mitotic cells were located in the inner enamel epithelium
and stratum intermediate, while the stellate reticulum

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ity even after recovery from solid-frozen human primary

tissue [39]. These findings suggest that cryopreserved
PDLSCs from extracted teeth could prove useful for clinically relevant therapeutic applications in the future.

Although it has been claimed that true mesenchymal

stem cells (MSCs) could be identified using a collection
of poorly defined markers [31], the resulting sorted cell
population still exhibits varying degrees of multipotentiality, indicative of heterogeneity. The fact that clonal cell
lines with a similar potential to regenerate bone in vivo
exhibit nonidentical marker expression profiles [32] is indicative of the fact that cultured DSC populations change
in culture, further confounding these studies. The task of
isolating purified dental MSC populations faces similar
difficulties to those encountered in generating purified
populations of other postnatal stem cell types, which also
lack suitable specific stem cell markers. However, the advantage with teeth is that they are one of the most accessible organs from which to derive stem cells, and DPSCs
can be cryopreserved while retaining their multipotential
differentiation capacity [33]. Therefore, dental MSCs can
likely provide an autologous population of neural crest
cell-derived stem cells for clinically relevant therapeutic
purposes in humans.
Another unique population of stem cells isolated from
human teeth is found at the tooth root apex. These cells
are called DSCs of the apical papilla (SCAP) and have
been demonstrated to differentiate into both odontoblasts and adipocytes. The higher proliferative potential
of SCAP as compared with DPSCs makes this population
of cells suitable for cell-based regeneration and preferentially for forming roots [34, 35].

Periodontal Tissue Regeneration

Periodontal tissue regeneration represents the ultimate goal of periodontal therapy and entails the formation of all components of the periodontium, including
gingiva, PDL, cementum and alveolar bone. In the early
1980s, attempts to regenerate periodontal tissues largely
focused on therapies designed to demineralize tooth root
cementum tissue in order to expose underlying collagen
fibers, with which newly formed collagen fibers could
subsequently integrate. This procedure often caused ankylosis and tooth root resorption, however, and thus the
procedure failed [42]. Another approach to periodontal
regeneration was the introduction of bone grafts into the
periodontal defect site. Although utilization of such
grafting materials for clinical repair of periodontal defects resulted in increased attachment levels of periodontal tissues and radiographic evidence of bone fill, histological assessment usually revealed that these materials
had little osteoinductive capacity and generally appeared
to be surrounded by dense fibrous connective tissue rather than organized periodontal tissue [42]. In recent years,
guided tissue regeneration has become the gold-standard
surgery for periodontal tissue regeneration. This procedure involves draping a biocompatible membrane over
the periodontal defect from the root surface to the adjacent alveolar bone, often in combination with a bone
graft. The barrier membrane prevents unwanted epithelium and gingival connective tissue from entering the
healing site, while promoting repopulation of the defect
site by cells migrating in from the PDL [38]. The rather
limited success of this approach has led scientists to develop methods to improve this therapy, through the addition of exogenous growth factors and via stem cell therapy.
Commonly used growth factors for PDL regeneration
therapies include bone morphogenetic proteins, plateletderived growth factor, Emdogain and recombinant amelogenin protein. The resultant improved regenerative
capability could be related to increased recruitment of
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progenitor MSCs, which subsequently differentiate to

form PDL tissue. Recently, PDLSCs transfected with expression vectors for platelet-derived growth factor and
bone morphogenetic protein were investigated in periodontal tissue engineering models [43, 44]. These studies
revealed the regeneration of normal periodontal tissues,
containing organized cementum, alveolar bone and the
PDL attachment apparatus. The possibility of constructing a root-periodontal tissue complex was further successfully demonstrated using a pelleted hydroxyapatite/
tricalcium phosphate scaffold containing SCAP, coated
with PDLSC-seeded Gelfoam, implanted and grown in
the minipig tooth socket [30].
The multipotent differentiation properties of PDLSCs
for generating both hard and soft tissues were further
demonstrated by constructing multilayered cell sheets
supported by woven polyglycolic acid. Transplanted cellseeded polyglycolic acid sheets regenerated new bone, cementum and well-oriented collagen fibers when inserted
into root surfaces. In addition to PDL-derived DSCs,
bone marrow-derived MSCs and adipose-derived stem
cells have been shown to promote periodontal tissue regeneration as well, although the mechanisms regulating
these processes have not been elucidated [44, 45]. One
hypothesis is that controlled MSC differentiation could
be developed as a therapeutically relevant approach for
PDL tissue regeneration. The combined use of transplanted MSCs and added exogenous signaling molecules
could accelerate the directed differentiation of MSCs in
vivo, providing more effective promotion of periodontal
tissue regeneration.
Successful therapies for PDL tissue regeneration will
not only facilitate the treatment of periodontal diseases,
but may also be used to improve current dental implant
therapies. Numerous attempts to reconstruct periodontal
tissues around dental implants revealed the challenge of
avoiding fibrous tissue encapsulation and the formation
of functional cementum on the implant surface [7].

Whole-Tooth Regeneration

Whole-tooth regeneration efforts largely consist of

two approaches: one involves in vivo implantation of immature tooth structures grown in vitro from dental progenitor cells, while the other uses in vitro expanded, cultured dental progenitor cell populations seeded onto
polymer scaffolds and implanted in vivo. Significant
progress in adult stem cell biology, biomaterials science
and the identification and characterization of DSCs have
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and outer enamel epithelium exhibited relatively reduced

proliferative activity. Recently, a specialized structure located at the apical region of the labial cervical loop in
mouse incisors was characterized and named the apical
bud [41]. Apical buds were suggested to act as epithelial
DSC-containing compartments that could differentiate
into ameloblasts through interaction with adjacent mesenchymal cells, including DPSCs.

to result in an improved ability to control tooth size and

shape. Dental mesenchymal cell-seeded polyglycolic acid
mesh draped with dental epithelial cell-seeded collagen
sponge gels that allowed for direct contact between dental
epithelial and mesenchymal cells were used to generate
organized tooth structures derived from dissociated canine and porcine molar tooth germs [51, 52]. In a similar
study, organized dentin-pulp complex, cementum and
PDL were shown to form in approximately one third of
experimental implants generated using gelatin-chondroitin-hyaluronan-tri-copolymer [53].
Although whole-tooth in vitro cell culture methods
are being further developed and improved, the task remains to identify available DSC populations to replace
the use of embryonic dental epithelial and mesenchymal
cells. The search for alternative sources of dental epithelium and mesenchyme is encouraged by the fact that dental epithelium can be created from non-teeth-bearing tissues and that non-dental, neural crest-cell derived mesenchyme can become competent for odontogenesis when
allowed to interact with inductive dental epithelium.
These experiments established the feasibility of instructing non-dental tissues to develop into teeth. Presumptive
DSCs such as DPSCs have been used to regenerate partial
teeth structures, but not an entire, functional biological
tooth. Due to the limited in vitro expansion abilities of
DPSCs, SHED and other DSC populations, significant efforts have been made to establish transgenic DSC lines
expressing transgenes, including human telomerase reverse transcriptase, SV40 T antigen and human papillomavirus genes, or through the identification of spontaneously immortalized dental follicle cells, Hertwigs epithelial root sheath, cementoblasts, dental papilla, PDL,
cervical loop epithelium, ameloblast and odontoblast lineage cells. One of the major technical advantages of this
approach is that cells can be produced, characterized and
controlled relatively easily without the need to derive material repeatedly from primary tissue. These lines could
therefore potentially be used to generate dental structures
in vivo.
A bigger challenge is to identify non-DSC populations
as potential replacements for dental epithelium and mesenchyme. It has been demonstrated that ESCs, neural
stem cells and bone marrow-derived cells can respond to
inductive signals from dental epithelium and express
odontogenic genes, and that bone marrow stromal cells
and dental epithelial recombinant explants can in fact
form tooth crowns composed of organized enamel, dentin, pulp and surrounding bone [54]. The teeth produced
were of the appropriate size and shape for mouse molars,

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contributed to recent successes in regenerating tooth

germs.
Early studies showed that it was possible to regenerate
tooth crowns from intact or partially dissected tooth
germs if suitable environments were provided, such as in
vitro organ culture, in vivo grafting on chick chorioallantoic membrane, ocular or subrenal grafts or subcutaneous transplants [4648]. Each of these implant sites
provides nutrients and oxygen to nurture tooth germ differentiation into mature teeth. Thus, there are several
choices for cultivating small tooth primordia prior to implantation into their anatomically relevant site in the jaw.
Optimally, the implant setting should reproduce an appropriate 3-dimensional organization for transplanted
cells to support functional differentiation, while avoiding
xenograft rejection. Therefore, organotypic culture is arguably the most relevant model system in which to grow
teeth in vitro.
By applying traditional tissue engineering methods,
tooth-like structures can be produced from biodegradable polymer scaffolds seeded with dissociated tooth
germ cells obtained from postnatal pigs or cultured rat
tooth bud cells grown in the omentum of immunocompromised mice [49]. Although harvested implants contained anatomically correct tooth crowns consisting of
organized enamel, dentin and pulp tissues closely resembling that of naturally formed teeth, they also contained
disorganized dental tissue and furthermore did not adopt
the size or shape of the biodegradable scaffold. Despite
these shortcomings, these results confirmed the ability of
mixed, heterogeneous populations of dental epithelial
and mesenchymal cells derived from dissociated adult
tooth germs to reaggregate within biodegradable scaffolds, interact and recapitulate odontogenic differentiation, to form adult tooth structures.
Examining the autonomous reaggregation capacity of
dissociated tooth germ cells further disclosed that cap
stage dental mesenchyme could induce dental epithelial
cell histogenesis even when positional memories have
been lost. Tooth morphogenesis of reaggregated tooth
germ cells showed characteristic developmental features
including the formation of functional odontoblasts and
ameloblasts, pulp and cusp formation and, following longer culture times, the formation of tooth roots and PDL
tissues [50]. These experiments demonstrated that cap
stage dental mesenchyme could control tooth cusp number and further reinforced the demonstrated ability for
mesenchymal and dental epithelium reaggregation.
Recently, it was found that dental cell-seeded tooth
scaffold constructs grown in a coculture system appeared

Current Challenges in Dental Tissue Engineering

A sufficient number of cell sources currently exist for

use in generating bioengineered dental mesenchymal tissue-derived tissues, including DPSCs, SHED, SCAP and
PDLSCs. In contrast, human dental epithelial stem cell
sources are limited for the following two reasons. Firstly,
dental epithelial cells undergo apoptosis after enamel formation is completed and therefore are no longer present
in erupted teeth. Undifferentiated wisdom or impacted
teeth are thus the only available sources of human dental
epithelium and are obtained either from children or
young adults. Secondly, ex vivo dental epithelial expansion can be difficult, due to the fact that it is inherently
more difficult to expand epithelial cells in culture as compared to mesenchymal cells. Therefore, in order to routinely bioengineer human whole teeth containing functional enamel, alternative dental epithelial cell sources
will have to be identified. Potential solutions to this problem are provided by studies demonstrating that bone
marrow-derived cells can give rise to ameloblast-like cells
[55], which indicates the potential use of non-dental cells
for enamel production.
It is anticipated that bioengineered tooth germs will be
implanted and grown in the jaw. Support for the feasibility of this approach is provided by recent reports demonstrating that implants placed in the naturally toothless
diastema in rodents and in both healed and fresh tooth
extraction sockets can accommodate tooth germ maturation in mice and canine jaws. The question that remains
is whether human jaws exhibit the same capacity. In addition to identifying the optimized development stage for
bioengineered tooth implantation, as well as the optimized implant placement procedure, proper tooth eruption is another important issue and concern. It is currently accepted that the dental follicle plays an important
role in the tooth eruption process, as indicated by published reports demonstrating that teeth lacking dental
follicles cannot erupt [56]. Teeth can erupt when a developing tooth is replaced by a silicon replica if the dental
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follicle is retained, but not when the follicle is not retained

[57]. Therefore, the creation of bioengineered tooth organs with an associated de novo dental follicle may likely
be the optimal design for successful tooth eruption.
Successful whole-tooth regeneration requires the formation of both functional tooth crown and root structures. To date, bioengineered tooth root formation with
accompanying functional PDL tissues has proved to be
quite challenging, with only a few reports of success [34],
indicating that increased effort will be needed to achieve
this goal. Finally, potential immune responses to bioengineered human dental implants have yet to be examined
and remain virtually unknown at this time. This important feature of biological replacement tooth therapies will
require careful attention prior to embarking on clinical
trials. Since it appears that human and mouse bone marrow stromal cells do not express costimulatory antigens,
DPSCs may similarly represent an immune privileged
population and not elicit humoral immune responses
[58]. Ideal tooth replacement therapies would use autologous cells harvested from the patient, thereby avoiding
potential immunological rejection responses.

Summary and Concluding Remarks

Dental tissue regeneration provides an attractive alternative to traditional, synthetic tooth restoration therapies. The hope is that patient-specific tissue-derived cell
populations can be used to functionally replace integral
tooth tissues. The development of such test tube teeth
requires precise regulation of the regenerative events in
order to achieve proper tooth size and shape, as well as
the development of new technologies to facilitate these
processes. In the future, it is anticipated that dental tissue
development and regeneration will exploit stem cells harvested from dental pulp or use non-DSC sources such as
iPS or induced DSCs. The practical use of DSCs for dental tissue engineering applications is at a primitive stage,
as current DSC sources are limited to specific developmental times. For example, dental follicle stem cells and
SCAP are available only during the wisdom tooth eruption stage in adolescence, while SHED/DPSCs or PDLSCs
can be harvested from exfoliated deciduous teeth, from
extracted wisdom teeth or from teeth extracted during
orthodontic treatment. Fortunately, it has been demonstrated that DSCs can be cryopreserved and used for
regenerative purposes at later times. Methods for constructing new tooth tissues include traditional recombination experiments, modified cell pellet cultures, scaffoldYen/Yelick

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reinforcing the idea that odontogenic signals could instruct tooth crown formation in embryonic or adult stem
cells without the use of a scaffold. Additional studies using c-kit-enriched bone marrow stromal cells demonstrated their ability to differentiate into ameloblast-like
cells [55], suggesting their potential to differentiate into
both dental epithelial and dental mesenchyme-derived
tissues.

based tissue engineering, assembly of bioengineered

tooth parts and gene therapy-based regeneration strategies. Several existing challenges in regenerative dentistry
still need to be overcome, including the need to establish
reliable ways to control tooth size, shape and color and to
create suitable jaw implantation site environments that
enable tooth development in vitro. Finally, methods to
control proper functional tooth eruption in adult jaws
must also be defined. Based on the current efforts, inter-

est and progress, it is tempting to speculate that clinically relevant bioengineered functional tooth therapies
for humans may be available in the near future. Although
this remains to be seen, it is nevertheless apparent that as
our knowledge and understanding of suitable methods
for successful dental tissue regeneration continue to increase it can be anticipated that these strategies will lead
to the significant benefits offered by biologically based
dental tissue replacement therapies.

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